Leptospirosis is a common zoonotic disease, typically transmitted to humans by rat exposure. Leptospiral infection in humans frequently results in fulminant, life-threatening illness characterized by liver dysfunction, kidney failure, and pulmonary hemorrhage. Leptospirosis is emerging in areas of the world undergoing rapid urbanization. Research is urgently needed to understand how these pathogenic spirochetes interact with their mammalian hosts. The focus of this proposal is to elucidate the molecular mechanisms of pathogenesis and immunity mediated by outer membrane proteins (OMPs). Our laboratory, a leader in this field, has developed strategies for membrane fractionation and identification of surface-exposed OMPs. We have identified a family of leptospiral immunoglobulin-like repeat (Lig) proteins that are targeted by the human immune response to leptospirosis. When virulent leptospires are exposed to host-physiologic levels of osmolarity (-300 mOsm/liter), expression of LigA and LigB is dramatically increased, while LipL36 and IPS expression are downregulated, changes known to be associated with infection of the mammalian host. Regulation by osmolarity indicates that Lig expression occurs early in the infectious process. We have recently shown that the LigA and LigB are fibronectin- and fibrinogen-binding proteins, indicating that these proteins are involved in host tissue colonization. In both LigA and LigB, the fibronectin- and fibrinogen-binding domains overlap, indicating a novel mechanism of protein-protein interaction. Specific Aim 1 describes studies designed to determine the role of Lig proteins in leptospiral binding to adhesive matrix molecules including fibronectin, fibrinogen, and laminin. We will examine the hypothesis that LigA and LigB are promiscuous adhesins able to bind multiple adhesive matrix molecules using a single adhesion domain. The goal of Specific Aim 2 is to identify osmo-upregulated OMPs exposed on the leptospiral surface during infection using immuno-histochemistry, reverse-transcriptase PCR, membrane fractionation, and surface-exposure analysis. Specific Aim 3 will determine which osmo-upregulated OMPs are protective immunogens in the hamster model of leptospirosis. We will examine the mechanism(s) by which antibodies to osmo-upregulated OMPs mediate protection using assays of bactericidal activity, growth inhibition, opsonophagocytosis, and adherence inhibition.